A major objective of this renewal is to determine what mechanisms control the emergence of diversity within categories of differentiated cells during embryonic development. When cells differentiate, they often form specific subsets of differentiated cells rather than generic cell types. While there are many examples of diversity within a cell type, an excellent example is vertebrate skeletal muscle cells. During avian and mammalian development, muscle fibers form in distinct generations and express disparate isoforms of myosin heavy chains (MyHCs). Fibers form in developmentally regulated, reproducible patterns of fiber distribution based on the controlled expression of slow isoforms of MyHC. Two processes, the mechanisms of which are not understood, control this diversification of myosin expression during embryogenesis - one is the innervation-independent generation of diverse, muscle-fiber precursors (myoblasts) and the other is the diversification of differentiated fibers by cell-cell interactions (innervation). To investigate these two mechanisms we cloned and sequenced a new gene for slow MyHC, slow MyHC 3, an embryonic form of the slow MyHC family and we developed cell culture models within which the control of myosin expression is dependent upon these two mechanisms. Slow MyHC 3 expression can be shown to be dependent on commitment of myoblasts to form slow fibers and by innervation of muscle fibers formed in cell culture. We will use this gene and the model systems to analyze: A) the formation of slow muscle fibers by nerve independent mechanisms that regulate slow MyHC 3 gene expression in fibers formed from defined myoblasts. Diverse fibers are first noted in muscle anlagen before limbs are innervated in mammals and birds. In this embryonic phase of myogenesis, slow MyHC 3 expression is independent of innervation. To study the, cell biology of this form of regulated expression we developed different types of embryonic myoblast cell lines from these anlagen, which when they differentiate into fibers in cell culture, either do or do not express slow MyHC 3. These myoblast lines will be used to characterize the cis-regulatory regions of the slow MyHC 3 gene responsible for slow fiber-type-specific expression. The purpose is to identify the mechanism(s) that permits some, but not all, differentiating myoblast types to express slow MyHCs, for it is this distinction that produces variations in muscle function in all vertebrates; and B) the formation of slow muscle fibers by nerve-dependent mechanisms that regulate slow MyHC 3 gene expression in neuron-muscle fiber co-culture. As early development proceeds another mechanism of fiber diversity emerges - innervation-dependent expression of slow myosin isoforms. To study this mechanism, we developed a cell-culture system wherein slow MyHC 3 gene expression in muscle cell cultures is dependent on innervation. The aim of these experiments is to determine the biological basis for innervation-dependent differentiation and identify the elements of the slow MyHC 3 gene responsive in selective activation of slow MyHC 3 by innervation.